Cystine diamide analogs for the prevention of cystine stone formation in cystinuria

ABSTRACT

Cystine analogs that improve the solubility of L-cystine in urine for treatment of cystinuria and which have the structure: 
     
       
         
         
             
             
         
       
     
     and pharmaceutically acceptable salts, solvates and prodrugs thereof, wherein
         each R and R′ pair are independently selected from (i) or (ii);   (i) R and R′ are independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alcohol, substituted or unsubstituted aryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclic, and substituted or unsubstituted heteroaryl, or   (ii) R and R′ together form a substituted or unsubstituted heterocyclic ring structure, or a substituted or unsubstituted heteroaryl ring structure;   X is hydrogen, or an alkyl; and Y is O or S.

CROSS-REFERENCE AND RELATED APPLICATION

This application claims priority under 35 U.S.C. §119 (e) to the U.S.Provisional Patent Application 61/748,323 filed on Jan. 2, 2013, thecontent and teachings of which are incorporated herein by reference inits entirety.

FIELD OF THE INVENTION

The present invention relates to novel cystine analogs, methods ofmaking cystine analogs, compositions containing cystine analogs andmethods of using such analogs for inhibiting cystine stone formation andtreatment of cystinuria. The cystine analogs of the present inventionimprove the aqueous solubility of L-cystine in vivo. The presentinvention also pertains to pharmaceutical compositions comprising thesenovel compounds for both diagnostic applications and treatment ofpathological cystinuria.

BACKGROUND OF THE INVENTION

Cystinuria is a rare chronic lifelong condition that affects about20,000 Americans. It is the result of an autosomal recessive disordercaused by mutations in one of the two genes, either SLC3A1 or SLC7A9,leading to abnormal transport of dibasic amino acids from the luminalfluid of the renal proximal tubules and small intestine. About 5% ofAmerican women and 12% of American men will develop a kidney stone atsome time in their lifetime, and prevalence has been rising in bothsexes.

Kidney stone recurrence is also common. It is estimated that almost 50%of stone formers will have a recurrence within 10 years. Approximately59% of kidney stones are calcium oxalate stones (pure) or with smallamounts of calcium phosphate; 10% are predominantly calcium phosphatestones; 17% are uric acid stones; 12% are struvite or infection stones;and remaining 2% are cystine and other stones. Although the rate ofcystine stones is much lower than calcium oxalate stones, cystine stonesare larger, recur more frequently, and are more likely to cause chronickidney disease. Medically, the disease caused by cystine stones in thekidney, ureter, and bladder is named cystinuria, which is a geneticabnormality results in abnormal transport of dibasic amino acids fromthe luminal fluid of the renal proximal tubules and small intestine.

Cystinuria is a chronic, lifelong condition and is most common in youngadults under age 40. It is the result of an autosomal recessive disordercaused by mutations in one of the two genes, either SLC3A1 on chromosome2 (type A) or SLC7A9 on chromosome 19 (type B), which code forcomponents of the major proximal renal tubule cystine and dibasic aminoacid transporter. Current clinical treatment of cystinuria aims toreduce the concentration of free cystine in urine and to increase itssolubility. A high fluid intake of around 4-5 liters a day andalkalinization of urine pH with citrate or bicarbonate salt can suppressbut may not completely prevent stone formation. At severe condition,chelation therapy is necessary, which utilizing the reaction ofD-penicillamine or α-mercaptopropionylglycine with L-cystine to generatemore soluble asymmetric disulfides. These drugs have side effectsincluding loss of taste, fever, proteinuria, serum sickness-typereactions, and even frank nephritic syndrome.

Recently, a group of researchers reported an alternative approach toprevent cystinuria based on crystal growth inhibition, which is achievedthrough the binding of tailored growth inhibitors-L-cystinedimethylester (CDME) and L-cystine methylester (CME) to specific crystalsurfaces. Real-time in situ atomic force microscopy (AFM) reveals thatCDME and CME dramatically reduce the growth velocity of sixsymmetry-equivalent {100} steps because of specific binding at thecrystal surface, which frustrates the attachment of L-cystine molecules.CDME almost completely inhibits the crystallization of L-cystine inwater with concentrations above 2 mg/L. While in cell cultureexperiments, CDME causes loss of cell viability at approximately 1 mM,and in rats study, demonstrates adverse effects at dosages ofapproximately 500 mg/kg per day.

Even though CDME inhibits cystine stone formation in the in vitro study,the methyl esters in CDME are cleavable by the variety of esteraseswidely present in almost all organs and tissues, most abundantly in ourdigestive system, blood and liver. In addition, the ester-mediatedhydrolysis of cystine esters will produce cystine, which would add tothe already elevated levels of cystine in the kidneys and bladder andpotentially increase the likelihood of cystine crystal formation andthus making the problem even worse. Therefore, there is a need for newand improved methods for treating cystinuria.

SUMMARY OF THE INVENTION

The present invention addresses the shortcomings in the prior artincluding those outlined above by providing cystine analog compoundswith enhanced ability to inhibit cystine crystal formations. The presentinvention provides new cystine analog compounds, methods of synthesizingsuch compounds, methods of evaluating stable analogs of cystine forinhibition cystine stone formation and methods for treating cystinuria.

In a first aspect, the present invention relates to diamide analogs ofL-cystine, and pharmaceutically acceptable salts, solvates, prodrugs andtautomers thereof. In this aspect of the invention, the cystine analogcompounds have the structure according to formula (A):

wherein each R and R′ pair are independently selected from (i) or (ii):

-   -   (i) R and R′ are independently selected from hydrogen,        substituted or unsubstituted alkyl, substituted or unsubstituted        alkenyl substituted or unsubstituted alcohol, substituted or        unsubstituted aryl, substituted or unsubstituted cycloalkyl,        substituted or unsubstituted heterocyclic, substituted or        unsubstituted heteroaryl, and    -   (ii) R and R′ together form a substituted or unsubstituted        heterocyclic, or a substituted or unsubstituted heteroaryl;        X is hydrogen, an alkyl, lower alcohol, and Y is O or S.

In one embodiment, with respect to formula (A), X is hydrogen and Y isO.

In one embodiment, with respect to formula (A), at least one R and R′pair together form a substituted or unsubstituted heterocyclic, or asubstituted or unsubstituted heteroaryl.

In at least another embodiment at least one R and R′ pair together forma ring structure having the formula (B):

wherein Z is CR″-Q, N-Q or O; and Q and R″ are independently selectedfrom hydrogen, substituted or unsubstituted alkyl, substituted orunsubstituted alkenyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, andsubstituted or unsubstituted cycloalkenyl, and r is 0-4.

In at least another embodiment, the structure (B) is selected from:

and Q is as defined above. In a preferred embodiment, the formed ringstructure is morpholine or piperazine.

In another aspect of the present invention, conjugates of formula (A)are prepared in the form of prodrugs designed to release one or more ofsuch compounds. In a preferred embodiment, the prodrugs of the presentcompounds have improved oral bioavailability and/or exhibit slower drugrelease in vivo.

In another embodiment, the disulfide bond is replaced by a linkerincluding substituted or unsubstituted lower alkylene chains such asethylene (—CH₂CH₂—), propylene (—CH₂CH₂CH₂—), methyleneoxy (—CH₂O—),ethyleneoxy (—CH₂CH₂O—), methyleneoxymethyl (—CH₂OCH₂—), methylenedioxy(—OCH₂O—), methylenesulfenyl (—CH₂S—), ethylenesulfenyl (—CH₂CH₂S—),methylenesulfenylmethyl (—CH₂SCH₂—), or like cycloalkyl rings such as1,4-cyclohexyl, 1,3-cyclopentyl, or like substituted cycloalkyl ringssuch as tetrahydropyran-2,5-diyl, tetrahydrofuran-2,5-diyl,tetrahydrothiophen-2,5-diyl, or substituted or unsubstituted aryl orheteroaryl rings.

Another aspect of the invention provides a pharmaceutical compositionfor inhibiting the formation of cystine kidney stone or slowing thegrowth of L-cystine crystallization comprising a pharmaceuticallyacceptable carrier and a pharmaceutically effective amount of a compoundaccording to formula (A).

Yet another aspect of the invention provides a method for treating,inhibiting or retarding the growth of L-cystine kidney-stone formationin a subject in need thereof, the method comprising administering to thesubject a pharmaceutically effective amount of a compound according toformula (A).

In yet another aspect of the invention provides a method of treating asubject having cystinuria, comprising administering to the subject inneed of such treatment a pharmaceutically effective amount of a compoundaccording to formula (A).

In a further aspect, the present invention provides pharmaceuticalcompositions, comprising a compound or compounds of the invention incombination with other modalities known in the art for treatment ofcystinuria, an acceptable pharmaceutical carrier, excipient or diluent.In this aspect of the invention, pharmaceutical composition can compriseone or more of the analog compounds described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a depiction of the effect of L-cystine diamindes on thesolubility of L-cystine in water and as compared to CDME.

FIGS. 2 (a)-(f) provide dose-response curves of five L-cystine diamidesin comparison to CDME. The figures show that the presently describeL-cystine diamides have equal or better activity than CDME at increasingthe aqueous solubility of L-cystine and inhibition of L-cystinecrystallization.

FIG. 3 provides the standard curve of cystine using the OPA method.

FIG. 4 provides a summary of effects of the novel compounds on cystinesolubility.

FIG. 5 provides pictures of stones found in mice treated.

FIG. 6 provides the drug concentration in mouse urine after 7 daily oraldosing of LH707 and LH708.

FIG. 7 provides the graph of the average compound concentration in mouseurine after 7 daily oral dosing of LH707 and LH708 in Slc3a1 knockoutmice and the control mice (the average and standard error were derivedfrom the 5 or 4 mice treated with LH707 and LH708, respectively).

FIG. 8 provides the table showing the volume of urine collected beforeand after treatment with LH707 and LH708.

FIG. 9 provides urine amino acid concentration upon a four-weektreatment with LH708. Accordingly, cystine was the only amino acid thathas significantly increased upon treatment with LH708 using theconventional criteria of statistical significance in paired t-test.

DETAILED DESCRIPTION OF THE INVENTION Definitions

When describing the compounds, pharmaceutical compositions containingsuch compounds and methods of using such compounds and compositions, thefollowing terms having the following meanings unless otherwiseindicated. It should also be understood that any of the moieties definedforth below may be substituted with a variety of substituents and thatthe respective definitions are intended to include such substitutedmoieties within their scope.

“Alkyl” refers to monovalent saturated alkane radical groupsparticularly having up to about 18 carbon atoms, more particularly as alower alkyl, from 1 to 8 carbon atoms and still more particularly, from1 to 6 carbon atoms. The hydrocarbon chain may be eitherstraight-chained or branched. This term is exemplified by groups such asmethyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl, tert-butyl,n-hexyl, n-octyl, tert-octyl and the like. The term “lower alkyl” refersto alkyl groups having 1 to 6 carbon atoms.

“Alkylene” refers to divalent saturated alkene radical groups having 1to 18 carbon atoms and more particularly 1 to 6 carbon atoms which canbe straight-chained or branched. This term is exemplified by groups suchas methylene (—CH₂—), ethylene (—CH₂CH₂—), and the like.

“Substituted alkyl” includes those groups recited in the definition of“substituted” herein, and particularly refers to an alkyl group having 1or more substituents, for instance from 1 to 5 substituents, andparticularly from 1 to 3 substituents, selected from acyl, acylamino,acyloxy, alkoxy, substituted alkoxy, alkoxycarbonyl,alkoxycarbonylamino, amino, substituted amino, aminocarbonyl,aminocarbonylamino, aminocarbonyloxy, aryl, aryloxy, azido, carboxyl,cyano, cycloalkyl, substituted cycloalkyl, halogen, hydroxyl,heteroaryl, keto, nitro, thioalkoxy, substituted thioalkoxy,thioaryloxy, thioketo, thiol, alkyl-S(O)—, aryl-S(O)—, and alkyl-S(O)₂—.

Substituted alkylene” includes those groups recited in the definition of“substituted” herein, and particularly refers to an alkylene grouphaving 1 or more substituents, for instance from 1 to 5 substituents,and particularly from 1 to 3 substituents, selected from acyl,acylamino, acyloxy, alkoxy, substituted alkoxy, alkoxycarbonyl,alkoxycarbonylamino, amino, substituted amino, aminocarbonyl,aminocarbonylamino, aminocarbonyloxy, aryl, aryloxy, azido, carboxyl,cyano, halogen, hydroxyl, keto, nitro, thioalkoxy, substitutedthioalkoxy, thioaryloxy, thioketo, thiol, alkyl-S(O)—, aryl-S(O)—, andalkyl-S(O)₂.

“Aryl” refers to a monovalent aromatic hydrocarbon group derived by theremoval of one hydrogen atom from a single carbon atom of a parentaromatic ring system. Typical aryl groups include, but are not limitedto, groups derived from aceanthrylene, acephenanthrylene, anthracene,azulene, benzene, fluoranthene, fluorene, hexacene, hexaphene, hexylene,as-indacene, s-indacene, indane, indene, naphthalene, octacene,octaphene, octalene, ovalene, penta-2,4-diene, pentacene, pentalene,pentaphene, perylene, phenalene, phenanthrene, picene, and the like.Particularly, an aryl group comprises from 6 to 14 carbon atoms.

Substituted “Aryl” includes those groups recited in the definition of“substituted” herein, and particularly refers to an aryl group that mayoptionally be substituted with 1 or more substituents, for instance from1 to 5 substituents, particularly 1 to 3 substituents, selected fromacyl, acylamino, acyloxy, alkenyl, substituted alkenyl, alkoxy,substituted alkoxy, alkoxycarbonyl, alkyl, substituted alkyl, alkynyl,substituted alkynyl, amino, substituted amino, aminocarbonyl,aminocarbonylamino, aminocarbonyloxy, aryl, aryloxy, azido, carboxyl,cyano, cycloalkyl, substituted cycloalkyl, halogen, hydroxyl, nitro,thioalkoxy, substituted thioalkoxy, thioaryloxy, thiol, alkyl-S(O)—,aryl-S(O)—, and alkyl-S(O)₂.

“Amino” refers to the radical —NH₂.

“Cycloalkyl” refers to cyclic hydrocarbyl groups having from 3 to about10 carbon atoms and having a single cyclic ring or multiple condensedrings, including fused and bridged ring systems, which optionally can besubstituted with from 1 to 3 alkyl groups. Such cycloalkyl groupsinclude, by way of example, single ring structures such as cyclopropyl,cyclobutyl, cyclopentyl, cyclooctyl, 1-methylcyclopropyl,2-methylcyclopentyl, 2-methylcyclooctyl, and the like, and multiple ringstructures such as adamantanyl, and the like.

“Substituted cycloalkyl” includes those groups recited in the definitionof “substituted” herein, and particularly refers to a cycloalkyl grouphaving 1 or more substituents, for instance from 1 to 5 substituents,and particularly from 1 to 3 substituents, selected from acyl,acylamino, acyloxy, alkoxy, substituted alkoxy, alkoxycarbonyl,alkoxycarbonylamino, amino, substituted amino, aminocarbonyl,aminocarbonylamino, aminocarbonyloxy, aryl, aryloxy, azido, carboxyl,cyano, cycloalkyl, substituted cycloalkyl, halogen, hydroxyl, keto,nitro, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioketo, thiol,alkyl-S(O)—, aryl-S(O)— and alkyl-S(O)₂.

“Hetero” when used to describe a compound or a group present on acompound means that one or more carbon atoms in the compound or grouphave been replaced by a nitrogen, oxygen, or sulfur heteroatom. Heteromay be applied to any of the hydrocarbyl groups described above such asalkyl, e.g. heteroalkyl, cycloheteroalkyl.

“Heteroaryl” refers to a monovalent heteroaromatic group derived by theremoval of one hydrogen atom from a single atom of a parentheteroaromatic ring system. Typical heteroaryl groups include, but arenot limited to, groups derived from acridine, carbazole, cinnoline,furan, imidazole, indazole, indole, indoline, indolizine, isobenzofuran,isochromene, isoindole, isoindoline, isoquinoline, isothiazole,isoxazole, naphthyridine, oxadiazole, oxazole, phenanthridine,phenanthroline, phenazine, phthalazine, pteridine, purine, pyran,pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole,pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline,tetrazole, thiadiazole, thiazole, thiophene, triazole, xanthene, and thelike. Preferably, the heteroaryl group is between 5-15 memberedheteroaryl, with 5-10 membered heteroaryl being particularly preferred.

“Pharmaceutically acceptable” means approved by a regulatory agency ofthe Federal or a state government or listed in the U.S. Pharmacopoeia orother generally recognized pharmacopoeia for use in animals, and moreparticularly in humans. As noted herein, the salts of the compounds ofthis invention refer to non-toxic “pharmaceutically acceptable salts.”Other salts may, however, be useful in the preparation of the compoundsaccording to the invention or of their pharmaceutically acceptablesalts.

“Pharmaceutically acceptable carrier” refers to a diluent, adjuvant,excipient or vehicle with which a compound of the invention isadministered.

“Prodrugs” refers to compounds, including derivatives of the compoundsof the invention, which have cleavable groups which respectively cleaveunder physiological conditions and form pharmaceutically active form ofthe present compounds in vivo. Such examples include, but are notlimited to, lower and long alkyl ester derivatives and the like, cholineand N-alkylmorpholine esters and the like. Prodrugs include acidderivatives well know to practitioners of the art, such as, for example,esters prepared by reaction of the parent acid with a suitable alcohol,or amides prepared by reaction of the parent acid compound with asubstituted or unsubstituted amine, or acid anhydrides, or mixedanhydrides. In some cases it is desirable to prepare double ester typeprodrugs such as (acyloxy)alkyl esters or((alkoxycarbonyl)oxy)alkylesters.

“Solvate” refers to forms of the compound that are associated with asolvent. Conventional solvents include water, ethanol, acetic acid andthe like. The compounds of the invention may be prepared e.g. incrystalline form and may be solvated or hydrated. Suitable solvatesinclude pharmaceutically acceptable solvates, such as hydrates, andfurther include both stoichiometric solvates and non-stoichiometricsolvates.

“Tautomers” refer to compounds that are interchangeable forms of aparticular compound structure, and that vary in the displacement ofhydrogen atoms and electrons. Thus, two structures may be in equilibriumthrough the movement of .pi. electrons and an atom (usually H). Forexample, enols and ketones are tautomers because they are rapidlyinterconverted by treatment with either acid or base. Another example oftautomerism is the aci- and nitro-forms of phenylnitromethane, that arelikewise formed by treatment with acid or base.

Tautomeric forms may be relevant to the attainment of the optimalchemical reactivity and biological activity of a compound of interest.

“Treating” or “treatment” of any disease or disorder refers, in oneembodiment, to ameliorating the disease or disorder (i.e., arresting orreducing the development of the disease or at least one of the clinicalsymptoms thereof). In another embodiment “treating” or “treatment”refers to ameliorating at least one physical parameter, which may not bediscernible by the subject. In yet another embodiment, “treating” or“treatment” refers to modulating the disease or disorder, eitherphysically, (e.g., stabilization of a discernible symptom),physiologically, (e.g., stabilization of a physical parameter), or both.In yet another embodiment, “treating” or “treatment” refers to delayingthe onset of the disease or disorder, or even preventing the same. In astill further embodiment, “treating” or “treatment” refers toadministration of the compound or composition of the invention forcosmetic purposes. “Prophylactic treatment” is to be construed as anymode of treatment that is used to prevent progression of the disease oris used for precautionary purpose for persons at risk of developing thecondition.

The compounds of the present invention may possess one or moreasymmetric centers; such compounds can therefore be produced asindividual (R)- or (S)-stereoisomers or as mixtures thereof.

As used herein, the EC_(2x)'s are the concentrations required of thecandidate compounds to double the aqueous solubility of cystine and weredetermined to assess drug potency and potential dosing regimens for invivo use. One of ordinary skill in the art is readily able to ascertainsuch information using commonly known methodologies.

For purposes of treating cystinuria in a mammalian subject, such as ahuman patient, an effective amount of one or more compounds of thepresent invention, or a pharmaceutically-acceptable salt thereof, isadministered to the subject to improve the solubility of cystine andprevent the formation or reduce the rate of growth of a cystine crystalsand kidney stones.

As discussed herein, the cystine analog compounds of the presentinvention can be administered in oral dosage forms such as tablets,capsules (each of which includes sustained release or timed releaseformulations), pills, powders, micronized compositions, granules,elixirs, tinctures, suspensions, syrups and emulsions. Likewise, theymay also be administered in intravenous (bolus or infusion),subcutaneous, intramuscular form, or other forms well known to those ofordinary skill in the pharmaceutical arts. For example, intramuscularinjection of a depot formulation containing lipophilic prodrugs in theform of long alkyl ester derivatives of the cystine analog compoundsmaybe used to slowly release the active cystine analog compounds forlong-term management of cystinuria. The ordinary skilled physician,veterinarian or clinician can readily determine and prescribe theeffective amount of the drug required to prevent, counter or arrest theprogress of the condition. Effective dosage forms, modes ofadministration and dosage amounts may be determined empirically and willvary with the activity of the particular compound employed, courseand/or progression of the disease state, the route of administration,the rate of excretion of the compound, renal and hepatic function of thepatient, the duration of the treatment, the identity of any other drugsbeing administered to the subject, age, size and like factors well knownin the medical arts.

Pharmaceutical formulations of the present invention include thosesuitable for oral, rectal, and/or parenteral administration. Regardlessof the route of administration selected, the active ingredient(s) areformulated into pharmaceutically-acceptable dosage forms by conventionalmethods known to those of skill in the art.

The amount of the active ingredient(s) which will be combined with acarrier material to produce a single dosage form will vary dependingupon the host being treated, the particular mode of administration andall of the other factors described above. The amount of the activeingredient(s) which will be combined with a carrier material to producea single dosage form will generally be that amount of the activeingredient(s) which is the lowest dose effective to produce atherapeutic effect.

Methods of preparing pharmaceutical formulations or compositions includethe step of bringing the active ingredient(s) into association with thecarrier and, optionally, one or more accessory ingredients. In general,the formulations are prepared by uniformly and intimately bringing theactive ingredient(s) into association with liquid carriers, or finelydivided solid carriers, or both, and then, if necessary, shaping theproduct.

Formulations of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or nonaqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and the like, each containing apredetermined amount of the active ingredient(s).

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules and the like), theprodrug(s), active ingredient(s) (in their micronized form) is/are mixedwith one or more pharmaceutically-acceptable carriers, such as sodiumcitrate or dicalcium phosphate, and/or any of the following: (1) fillersor extenders, such as starches, lactose, sucrose, glucose, mannitol,and/or silicic acid; (2) binders, such as, for example,carboxymethyl-cellulose, alginates, gelatin, polyvinyl pyrrolidone,sucrose and/or acacia; (3) humectants, such as glycerol; (4)disintegrating agents, such as agar-agar, calcium carbonate, potato ortapioca starch, alginic acid, certain silicates, and sodium carbonate;(5) solution retarding agents, such as paraffin; (6) absorptionaccelerators, such as quaternary ammonium compounds; (7) wetting agents,such as, for example, cetyl alcohol and glycerol monostearate; (8)absorbents, such as kaolin and bentonite clay; (9) lubricants, such astalc, calcium stearate, magnesium stearate, solid polyethylene glycols,sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents.In the case of capsules, tablets and pills, the pharmaceuticalcompositions may also comprise buffering agents. Solid compositions of asimilar type may also be employed as fillers in soft and hard-filledgelatin capsules using such excipients as lactose or milk sugars, aswell as high molecular weight polyethylene glycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered activeingredient(s) moistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient(s) thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter. These compositions mayalso optionally contain opacifying agents and may be of a compositionthat they release the active ingredient(s) only, or preferentially, in acertain portion of the gastrointestinal tract, optionally, in a delayedmanner. Examples of embedding compositions which can be used includepolymeric substances and waxes. The active ingredient(s) can also be inmicroencapsulated form.

Liquid dosage forms for oral administration of the active ingredient(s)include pharmaceutically-acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient(s), the liquid dosage forms may contain inert diluentscommonly used in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethylacetate, butyl alcohol, benzyl benzoate, propylene glycol,glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive,castor and sesame oils), glycerol, amyl alcohol, tetrahydrofurylpolyethylene glycols and fatty acid esters of sorbitan, and mixturesthereof.

Besides inert diluents the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents. Suspensions, inaddition to the active ingredient(s), may contain suspending agents as,for example, ethoxylated alcohols, polyoxyethylene sorbitol and sorbitanesters, microcrystalline cellulose, aluminum metahydroxide, bentonite,agar-agar and tragacanth, and mixtures thereof.

Oral dosages of the present invention, when used for the indicatedeffects, will range between about 0.01 mg per kg of body weight per day(mg/kg/day) to about 5000 mg/kg/day, preferably 0.1 to 2500 mg/kg/day,and most preferably 10 to 1500.0 mg/kg/day. For oral administration, thecompositions are preferably provided in the form of tablets containing0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 500and 1000 milligrams of the cystine diamide analog compounds of thepresent invention ingredient for the symptomatic adjustment of thedosage to the patient to be treated.

The compounds of the present invention can also be administered in theform of liposome delivery systems, such as small unilamellar vesicles,large unilamellar vesicles and multilamellar vesicles. Liposomes can beformed from a variety of phospholipids, such as cholesterol,stearylamine or phosphatidylcholines.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise the active ingredient(s) or prodrug form(s) incombination with one or more pharmaceutically-acceptable sterileisotonic aqueous or nonaqueous solutions, suspensions or emulsions, orsterile powders which may be reconstituted into sterile injectablesolutions or dispersions just prior to use, which may containantioxidants, buffers, solutes which render the formulation isotonicwith the blood of the intended recipient or suspending or thickeningagents.

Injectable depot forms are made by forming microencapsule matrices ofthe active ingredient(s) or prodrug form(s) in biodegradable polymerssuch as polylactide-polyglycolide. Depending on the ratio of the activeingredient(s) to polymer, and the nature of the particular polymeremployed, the rate of release of the active ingredient(s) can becontrolled. Examples of other biodegradable polymers includepoly(orthoesters) and poly(anhydrides). Depot injectable formulationsare also prepared by entrapping the active ingredient(s) in liposomes ormicroemulsions which are compatible with body tissue. The injectablematerials can be sterilized for example, by filtration through abacterial-retaining filter.

Another aspect of the present invention is directed to methods oftreating a subject diagnosed with cystinuria by administering to saidsubject a pharmaceutically effective amount of the presently disclosedcompounds. As noted herein, the cystine analog compounds of the presentinvention can be used in combination with other agents used fortreatment of cystinuria or other agents which will enhance suchtreatment regime for subjects in need of such treatment. The individualcomponents of such combinations can be administered separately atdifferent times during the course of therapy or concurrently in dividedor single combination forms to patients or regions of such patients inneed of such therapy. The instant invention is therefore to beunderstood as embracing all such regimes of simultaneous or alternatingtreatment and the term “administering” is to be interpreted accordingly.It will be understood that the scope of combinations of the compounds ofthis invention with other agents useful to treat the targeted conditionincludes in principle any combination with any pharmaceuticalcomposition useful for treating disorders related to kidney stone orrelated chronic kidney disease.

In at least one aspect of the present invention, it is more convenientor desirable to prepare, purify, and/or handle the active compound inthe form of a prodrug. The term “prodrug” as used herein, pertains to acompound which, when metabolized, yields the desired active compound orin itself is the active compound. This includes for example adding aphosphoric acid ester moiety, alkoxycarbonyl (ROCO),(acyloxy)alkoxycarbonyl (RCOOCH(R′) OCO, where R and R′ are the same asabove; in suitable positions such as positions X or Q in formulas A andB. Typically, the prodrug is inactive, or less active than the activecompound, but may provide advantageous handling, administration, ormetabolic properties.

Various cystine diamide analogs in accordance with this invention wouldbe understood by those skilled in the art made aware of this invention,as available through synthetic procedures of the sort described hereinor straight-forward modifications thereof.

At least one aspect of the present invention provides for methods ofsynthesizing the present compounds and further preparing suitableformulations. In at least one embodiment, the method of synthesizingcysteine diamide analog follows the steps of: (i) obtaining N-alkylcysteine by reducing thiazolidine-4-carboxylic acid; (ii) producing N,N′ dialkyl cysteine by oxidizing the N-alkyl cysteine of step (i) in thepresence of suitable transitional metal catalytic compound; (iii)protecting the secondary amine with Boc anhydride, and amidation throughactivated ester; and (iv) deprotecting the target N,N′-substitutedL-cystine.

In more specific embodiment, the compounds of the present invention canbe synthesized by the following methods as depicted in the schemesbelow:

Scheme 1 provides the making of compounds I-IX through the amidation ofL-cystine using activated OSu or OBt ester and subsequent deprotectionof Boc group using 50% TFA in CH₂Cl₂ or 4 equiv. of 4 N HCl in dioxane.Amidation using activated esters was found to give better reactionyields and fewer side products. The total yields of the three stepsequence ranged from 10% to 50%.

Several conditions were initially used to direct methylate L-cystine butfailed to obtain N,N′-dimethyl L-cystine. The process was thus modifiedaccording to Scheme 2 to first obtain N-methyl cysteine through Na—NH₃reduction of thiazolidine-4-carboxylic acid and then air oxidize theN-methyl cysteine in the presence of catalytic iron (II) chloride toafford the desired N,N′-dimethyl L-cystine. Protection of the secondaryamine with Boc anhydride, amidation through activated ester andsubsequent deprotection afford the target N,N′-dimethyl L-cystine amidesX-XVI in 10% to 30% overall yield

As described herein, the cystine analogs of the present invention havethe structure formula (A):

wherein each R and R′ pair are independently selected from (i) or (ii)

-   -   (i) R and R′ are independently selected from hydrogen,        substituted or unsubstituted alkyl, substituted or unsubstituted        alkenyl, substituted or unsubstituted alcohol, substituted or        unsubstituted aryl, substituted or unsubstituted cycloalkyl,        substituted or unsubstituted heterocyclic, substituted or        unsubstituted heteroaryl, and    -   (ii) R and R′ together form a substituted or unsubstituted        heterocyclic ring structure, or a substituted or unsubstituted        heteroaryl ring structure; and

X is hydrogen, an alkyl, lower alcohol, and Y is O or S.

In one embodiment, at least one R and R pair together form a ringstructure having the formula (B):

wherein Z is CR″-Q, N-Q or O; and Q and R″ are independently selectedfrom hydrogen, substituted or unsubstituted alkyl, substituted orunsubstituted alkenyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, andsubstituted or unsubstituted cycloalkenyl, and r is 0-4.

In at least another embodiment, the structure (B) is selected from:

and Q is as defined above. In a preferred embodiment the formed ring ismorpholine or piperazine.

In at least one embodiment with respect to formula (A), the cystineanalog compound having the structures according to the followingformulas:

In a more preferred embodiment the compounds have a structure accordingto the following formulas:

At least one aspect of the present invention is to provide series ofcystine diamides that increases the aqueous solubility of L-cystine. Inat least one embodiment, the cysteine concentration in urine of asubject receiving the cysteine diamide increases by at least 30%, 40%,50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%. In a preferred embodiment, theurine concentration of cysteine is increased by at least 70%, while theconcentration of other urine amino acids remains statistically unchangedafter the completion of the treatment regimen.

Another aspect of the present invention introduces cystine diamides thatexhibits a 1-1000 fold increase in potency as compared to thoseavailable in the prior art. At least in another embodiment the compoundsof the present invention show an increase in potency by 7 to 50 folds.At least two compounds of the present invention exhibited at least a 7and a 24 fold increase in potency as compared to the control CDME. Thepresent compounds having the formulas VII and VIII respectively showed a7.44 and 24.41 fold increase in activity as compared to CDME.

In at least another aspect of the present invention, a prodrug conjugateis designed according to the structure of formula (C):

wherein each R and R′ pair are independently selected from (i) or (ii):

-   -   (i) R and R′ are independently selected from hydrogen,        substituted or unsubstituted alkyl, substituted or unsubstituted        alkenyl, substituted or unsubstituted alcohol, substituted or        unsubstituted aryl, substituted or unsubstituted cycloalkyl,        substituted or unsubstituted heterocyclic, substituted or        unsubstituted heteroaryl, and    -   (ii) R and R together form a substituted or unsubstituted        heterocyclic ring structure, or a substituted or unsubstituted        heteroaryl ring structure;

Y is O or S; and X′ is selected from alkoxycarbonyl (ROCO),(acyloxy)alkoxycarbonyl (RCOOCH(R′)OCO, where R and R′ are the same asabove.

In at least another embodiment of this aspect of the invention, at leastone of the R and R′ of the prodrug conjugate form a ring structurehaving the formula (D):

wherein Z′ is CR″—W, N—W or O; and R″ is selected from hydrogen,substituted or unsubstituted alkyl, substituted or unsubstitutedalkenyl, substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted cycloalkyl, and substituted orunsubstituted cycloalkenyl, and W is selected from alkoxycarbonyl(ROCO), (acyloxy)alkoxycarbonyl (RCOOCH(R′)OCO, where R and R′ are thesame as above; and r is 0-4.

As set forth herein the prodrugs of the disclosed compounds are designedto release one or more of such compounds. In yet another embodiment, theprodrug has the general formula of (C) and (D) as above, wherein X′and/or W is selected from alkoxycarbonyl (ROCO), (acyloxy)alkoxycarbonyl(RCOOCH(R′)OCO, R and R′ are the same as above.

In another embodiment, the disulfide bond is replaced by a linker.Accordingly, the cystine analogs of the present invention can have thestructure formula (E):

wherein each T and T′ pair are independently selected from (i) or (ii):

-   -   (i) T and T′ are independently selected from hydrogen,        substituted or unsubstituted alkyl, substituted or unsubstituted        alkenyl, substituted or unsubstituted alcohol, substituted or        unsubstituted aryl, substituted or unsubstituted cycloalkyl,        substituted or unsubstituted heterocyclic, substituted or        unsubstituted heteroaryl, and    -   (ii) T and T′ together form a substituted or unsubstituted        heterocyclic ring structure, or a substituted or unsubstituted        heteroaryl ring structure;

X″ is hydrogen, an alkyl, lower alcohol, and Y″ is O or S; and

L is a linker selected from a group consisting of substituted orunsubstituted lower alkylene chains such as ethylene (—CH₂CH₂—),propylene (—CH₂CH₂CH₂—), methyleneoxy (—CH₂O—), ethyleneoxy (—CH₂CH₂O—),methyleneoxymethyl (—CH₂OCH₂—), methylenedioxy (—OCH₂O—),methylenesulfenyl (—CH₂S—), ethylenesulfenyl (—CH₂CH₂S—),methylenesulfenylmethyl (—CH₂SCH₂—), or substituted or unsubstitutedcycloalkyl rings such as 1,4-cyclohexyl, 1,3-cyclopentyl,tetrahydropyran-2,5-diyl, tetrahydrofuran-2,5-diyl,tetrahydrothiophen-2,5-diyl, or substituted or unsubstituted aryl orheteroaryl rings.

In one embodiment, at least one T and T′ pair together form a ringstructure having the formula (F):

wherein Z′ is CT″-Q′, N-Q′ or O; and Q′ and T″ are independentlyselected from hydrogen, substituted or unsubstituted alkyl substitutedor unsubstituted alkenyl, substituted or unsubstituted aryl, substitutedor unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl,and substituted or unsubstituted cycloalkenyl, and p is 0-4.

In at least another embodiment, the structure (F) is selected from:

and Q′ is as defined above. In a preferred embodiment the formed ring ismorpholine or piperazine.

The general methods given in Schemes 1 and 2 for the preparation ofcystine analog compounds disclosed here and further in Table I. Thepreferred cystine analog compounds of the present invention are furtherillustrated by the following examples. Unless otherwise specified allstarting materials and reagents are of standard commercial grade, andare used without further purification, or are readily prepared from suchmaterials by routine methods. Those skilled in the art of organicsynthesis will recognize that starting materials and reaction conditionsmay be varied to achieve the desired end product.

EXAMPLES Example 1 Experimental Synthesis of L-Cystine Diamides (I-IX)

NHS (1.7 g, 15.0 mmol) and DCC (3.1 g, 15.0 mmol) were added thesolution of N,N′-bis(tert-butoxycarbonyl)-L-cystine (3.0 g, 6.8 mmol) inethyl acetate (200 mL), and the mixture was stirred at r.t. for 2 hr.After the filtration, the liquid was concentrated and dissolved inanhydrous DCM. After filtration to remove the DCU precipitates, thesolvent was removed under reduced pressure to give the crude activatedester as a white solid (2.1 g, 48.6%), which was used without furtherpurification. Similarly, HOBt and EDC can be used to prepareN,N′-Bis(tert-butoxycarbonyl)-L-cystine OBt ester for reaction withamines.

The activated esters of N,N′-Bis(tert-butoxycarbonyl)-L-cystine weredissolved in a suitable solvent such as acetonitrile, methylenechloride, DMF or NMP. To the solution of an activated ester ofN,N′-Bis(tert-butoxycarbonyl)-L-cystine, excess amine (3-10 eq) wasadded and the reaction was allowed to proceed at r.t. for 2 hr. At theend of reaction as monitored by LC-MS or TLC, solvent was removed underreduced pressure and the residue was dissolved in DCM and washed withMillipore water for 3 times. After concentration, the crude product waspurified by ISCO (normal phase, DCM-20% MeOH/DCM). The purified oil wasdissolved into 50% TFA/DCM and stirred for 1 hr to remove the Bocprotecting group. After TFA and DCM were removed by N₂, diethyl etherwas used to precipitate desired product. After centrifuge, the solid wascollected and dissolved into Millipore water, and washed with ethylacetate for 3 times. The aqueous solution was lypholized to finalproduct. To obtain the HCl salt of L-cystine diamides, deprotection ofBoc-cystine diamides was performed using 4 N HCl in dioxane (4 equiv).

L-Cystine diamide (CDAA, LH701, I): 8.1 mg (71.9% yield). LC-MS (ESI+)m/z 238.9 [M+H]⁺; 1H-NMR (400 MHz, D₂O, δ): 4.24 (dd, 2H), 3.28 (dd,2H), 3.07 (dd, 2H).

L-Cystine bis(dimethylamide) (CDMA, LH702, II): 4.2 mg (30.2% yield).LC-MS (ESI+) m/z 294.9 [M+H]⁺; 1H-NMR (400 MHz, D₂O, δ): 4.78 (m, 2H),3.30 (dd, 2H), 3.12 (dd, 2H), 3.10 (s, 6H), 2.93 (s, 6H).

L-Cystine bis(diethylamide) (CDEA, LH703, II): 3.7 mg (22.3% yield).LC-MS (ESI+) m/z 350.8 [M+H]⁺; 1H-NMR (400 MHz, D₂O, δ): 4.78 (m, 2H),3.46 (m, 6H), 3.29 (m, 4H), 3.20 (m, 2H), 1.20 (t, 6H), 1.09 (t, 6H).

L-Cystine bis(cyclopropylamide) (CDCPA, LH704, IV): 8.6 mg (57.2%yield). LC-MS (ESI+) m/z 318.8 [M+H]⁺; 1H-NMR (400 MHz, D₂O, δ): 4.13(t, 2H), 3.20 (dd, 2H), 3.11 (dd, 2H), 2.57 (p, 2H), 0.72 (m, 4H), 0.50(m, 4H).

L-Cystine bis(pyrrolidine) (CDPYR, LH705, V): 12.6 mg (76.9% yield).LC-MS (ESI+) m/z 346.9 [M+H]⁺; 1H-NMR (400 MHz, D₂O, δ): 4.57 (dd, 2H),3.61 (m 2H), 3.54 (m, 2H), 3.46 (m, 2H), 3.36 (m, 2H), 3.29 (dd, 2H),3.14 (dd, 21H), 1.95 (m, 4H), 1.86 (m, 4H).

L-Cystine bispiperidine (CDPIP, LH706, VI): 11.5 mg (65.0 yield). LC-MS(ESI+) m/z 374.8 [M+H]⁺; 1H-NMR (400 MHz, D₂O, δ): 4.78 (m, 2H), 3.46(t, 8H), 3.16 (m, 4H), 1.57 (m, 6H), 1.50 (m, 6H).

L-Cystine bis(N′-methylpiperazide) (CDNMP, LH708, VIII): 12.8 mg (67.0%yield). LC-MS (ESI+) m/z 404.9 [M+H]⁺; 1H-NMR (400 MHz, D₂O, δ): 4.76(m, 2H), 3.56˜3.78 (m, 22H), 3.24 (m, 4H).

L-Cystine bismorpholide (CDMOR, LH707, VII): 10.2 mg (57.0% yield).LC-MS (ESI+) m/z 378.8 [M+H]⁺; 1H-NMR (400 MHz, D₂O, δ): 4.80 (t, 2H),3.60˜3.76 (m, 16H), 3.25 (m, 4H).

L-Cystine diethanolamide (CDEOA, LH709, IX): 6.3 mg (40.8% yield). LC-MS(ESI+) m/z 326.8 [M+H]⁺; 1H-NMR (400 MHz, D₂O, δ): 4.18 (t, 2H), 3.66(t, 4H), 3.24 (m, 6H), 2.99 (m, 2H).

Example 2 Synthesis of N,N′-dimethyl L-cystine diamides (X-XVI)

Thiazolidine-4-carboxylic acid (6.66 g, 50 mmol) was dissolved in 60 mLof liquid ammonia at −78° C., to which was added H₂O (0.9 mL). Solidsodium was added until the solution remained blue (˜3.8 g). The reactionwas quenched with the addition of NH₄Cl (11 g, 205.6 mmol), after whichthe mixture was allowed to warm to room temperature and evaporateovernight. After drying under vacuum, the crude white solid wasdissolved in 50 mL of water and acidified to pH 1 with 6 N HCl. Thesolvent and excess acid were removed under reduced pressure to reveal asticky off-white solid. The solid was extracted with absolute EtOH,which yielded a sticky yellow solid after removal of the solvent. TheN-methylated cysteine was air-oxidized to the disulfide by dissolving in250 mL of water, at pH 9 (adjusted with ammonium hydroxide), in presenceof iron (II) chloride (1 crystal) and bubbling with air overnight. After13 h, the solution tested negative for thiolates using the nitroprussidereaction. The solution was acidified to pH 6 with 25% AcOH. Addition of100 mL of absolute EtOH precipitated a thick white solid which wasremoved by centrifugation and washed twice with 50 EtOH. The solid wasdissolved in water and lyophilized yielding 4.3 g (32.1% yield) ofN,N′-dimethyl cystine as fluffy off-white solid. LC-MS (ESI+): m/z 268.7[M+H]⁺.

To a stirred solution of N,N′-dimethyl L-cystine (2.5 g, 9.32 mmol) insodium bicarbonate solution (50 mL) at 0° C., was added di-tert-butyldicarbonate (6.0 g, 27.4 mmol) in acetone (10 mL) dropwise over thecourse of approximately 2 hr. The reaction was stirred overnight andallowed to slowly warm to r.t. The pH of reaction mixture was adjustedto 5 using 5% sodium bisulfate solution The mixture was extracted withethyl acetate (3×100 mL). The combined organic layers were dried overNa₂SO₄ and concentrated in vacuo. The residue was purified by columnchromatography on silica gel to yield 2.3 g (51.8% yield) ofN,N′-Bis(tert-butoxycarbonyl)-N,N′-dimethyl L-cystine. LC-MS (ESI+): m/z468.9 [M+H]⁺.

The N,N′-dimethyl L-cystine diamides (X-XVI) were prepared in a similarfashion as above by activatingN,N′-Bis(tert-butoxycarbonyl)-N,N′-dimethyl L-cystine with HOSu/DCC orHOBt/EDC, subsequently reacting the activated esters with excess amines,and final deprotection with 50% TFA/CH₂Cl₂ or 4 N HCl in dioxane.

N,N′-Dimethyl L-cystine bis(dimethylamide) (Me-CDMA, LH710, X): 6.2 mg(42.5% yield). LC-MS (ESI+) m/z 322.9 [M+H]⁺; 1H-NMR (400 MHz, D₂O, δ):4.70 (m, 2H), 3.30 (t, 4H), 3.11 (s, 6H), 2.95 (s, 6H), 2.67 (s, 6H).

N,N′-Dimethyl L-cystine bis(diethylamide) (Me-CDEA, LH711, XI): 2.3 mg(13.4% yield). LC-MS (ESI+) m/z 378.8 [M+H]⁺; 1H-NMR (400 MHz, D₂O, δ):4.60 (m, 2H), 3.29˜3.50 (m, 12H), 2.68 (s, 6H), 1.21 (t, 6H), 1.11 (t,6H).

N,N′-Dimethyl L-cystine bis(cyclopropylamide) (Me-CDCPA, LH712, XII):8.8 mg (56.1% yield). LC-MS (ESI+) m/z 346.8 [M+H]⁺; 1H-NMR (400 MHz,D₂O, δ): 4.01 (t, 2H), 3.23 (m, 4H), 2.67 (s, 6H), 2.66 (m, 2H), 0.76(m, 4H), 0.54 (m, 4H).

N,N′-Dimethyl L-cystine bis(pyrrolidine) (Me-CDPYR, LH713, XIII): 7.6 mg(44.7% yield). LC-MS (ESI+) m/z 374.8 [M+H]⁺; 1H-NMR (400 MHz, D₂O, δ):4.48 (t, 2H), 3.64 (m, 2H), 3.55 (m, 2H), 3.48 (m, 2H), 3.40 (m, 2H),3.29 (dd, 4H), 2.68 (s, 6H), 1.95 (m, 4H), 1.88 (m, 4H).

N,N′-Dimethyl L-cystine bispiperidine (Me-CDPIP, LH714, XIV): 9.4 mg(51.6% yield). LC-MS (ESI+) m/z 402.8 [M+H]⁺; 1H-NMR (400 MHz, D₂O, δ):4.78 (m, 2H), 3.52 (m, 8H), 3.29 (d, 4H), 2.67 (s, 6H), 1.62 (m, 6H),1.56 (m, 6H).

N,N′-Dimethyl L-cystine bismorpholide (Me-CDMOR, LH715, XV): 5.1 mg(27.7% yield). LC-MS (ESI+) m/z 406.8 [M+H]⁺; 1H-NMR (400 MHz, D₂O, δ):4.70 (m, 2H), 3.73 (m, 8H), 3.62 (m, 8H), 3.30 (t, 4H), 2.68 (s, 6H).

N,N′-Dimethyl L-cystine diethanolamide (Me-CDEOA, LH716, XVI): 4.8 mg(30.1% yield). LC-MS (ESI+) m/z 354.8 [M+H]⁺; 1H-NMR (400 MHz, D₂O, δ):4.15 (t, 2H), 3.65 (m, 4H), 3.42 (m, 4H), 3.29 (d, 4H), 2.68 (s, 6H).

Another aspect of the present invention is directed to the processes ofdetermining the concentration of L-cystine using a fluorescence assayafter OPA/NBC derivatization. In at least one embodiment with respect tothis aspect of the invention, the concentration of the L-cystine wasassessed by a fluorescence assay. Accordingly, the inventors developed afluorescence-based assay using O-phthaldialdehyde (OPA) andN-Boc-cysteine (NBC) to accurately measure the concentration ofL-cystine in aqueous solutions using a similar protocol we previouslyreported. OPA/NBC are popular reagents used for precolumn derivatizationof amino acids in HPLC. This derivatization is fast and simple tooperate. However, the reaction of OPA with the disulfide cystine and NBCyielded derivatives with weak fluorescence. The disulfide in cystine wasreduced and then alkylated with iodoacetic acid to formS-carboxymethylcysteine, which yielded OPA/NBS derivatives with normalfluorescence. L-Cystine solution was thus used to construct a standardcurve. L-Cystine first reacted with DTT for 10 min, followed by reactionwith iodoacetic acid for 15 min, then reacted with OPA and NBC for 3min. The final mixture was pipetted into 384-well plate and thefluorescence intensity was read at Ex 340 nm/Em 460 nm. A linearstandard curve was obtained in the concentration range between 50 μM and400 μM (see FIG. 3).

Example 3 Fluorescence Assay for Inhibition of L-Cystine CrystalFormation

Formation of supersaturation solution—a supersaturated solution wasformed by dissolving 21 mg of L-cystine in 30 mL of Millipore water (˜3mM) under reflux at 100° C. for 20 min until the L-cystine wascompletely dissolved. The supersaturated solution was then allowed tocool slowly with stirring for 75 min.

Construction of standard curves—L-Cystine (5 mg) was dissolved inMillipore water (34.7 mL) to form a 0.6 mM solution as a stock solution.Then, L-cystine solution was diluted to 0.4, 0.3, 0.2, 0.1, 0.05 mMsolution. 10 uL of each L-cystine solutions, 90 uL of 0.1 M dibasicsodium phosphate solution, and 10 uL of DTT solution (12.5 mM) weremixed at r.t. for 10 min. before the addition of 10 uL of iodoaceticacid (100 mM) and continued incubation at r.t. for an additional 15 min.This was then followed by the addition of 10 uL of OPA (100 mM inmethanol) and 10 uL of NBC (100 mM in methanol). The derivatization wasallowed to proceed for 3 min before 40 uL of the mixture was plated in a384-well plate and read at Ex 340 nm/Em 460 nm. The standard curve wasrepeated for each set of experiments and used to calculate theconcentration of L-cystine in each sample.

The mechanism of OPA method for determination of cystine concentrationis depicted in the following scheme:

Determination of L-cystine concentration—All test compounds weredissolved in water to form 10 mM stock solution. 5 uL of each solutionwas added to 500 uL L-cystine supersaturated solution. The mixtures wereallowed to stand at 25° C. for 72 h. At the end of incubation, themixtures were centrifuged at 10,000 rpm for 4 min and the supernatantswere diluted 2-fold for concentration measurement. Each diluted mixture(10 uL), 0.1 M dibasic sodium phosphate solution (90 uL), and 10 uL ofDTT solution (12.5 mM) were mixed at r.t. for 10 min, before theaddition of 10 uL of iodoacetic acid (100 mM) and continued incubationat r.t. for an additional 15 min. Derivatization was performed by theaddition of 10 uL of OPA (100 mM in methanol) and 10 uL of NBC (100 mMin methanol) for 3 min. 40 uL of the derivatized mixture was plated in a384-well plate and fluorescence was read at Ex 340 nm/Em 460 nm toderive the concentrations of the original mixtures.

Example 4 Primary Screening of L-Cystine Diamides Synthesized

In order to determine the effects of L-cystine diamides synthesized onthe aqueous solubility of L-cystine, a supersaturated solution ofL-cystine was prepared in Millipore water according to the literaturemethod. Then, 1 mM and 200 uM solutions of each L-cystine diamidesynthesized were added to a supersaturated solution of L-cystine inwater (1:100) to give supersaturated solutions of L-cystine containing10 μM and 2 μM of a L-cystine diamide, respectively. The mixtures werethen allowed to incubate at 25° C. for 72 h; the solubility of L-cystinein the presence of 10 μM and 2 μM of each compound were determined usingthe above fluorescence assay (see FIG. 4). CDME was used as a positivecontrol.

As shown in FIG. 1, L-cystine diamides of the present invention havebetter activity than CDME at increasing the aqueous solubility ofL-cystine and thus inhibiting L-cystine crystallization while thecorresponding methylated analogs, N,N′-dimethyl L-cystine diamides, havelittle effect on the aqueous solubility of L-cystine. The failure ofN,N′-dimethyl L-cystine diamides to inhibit crystallization of L-cystinecould be due to the fact that the N-methyl substituent adverselyaffected intermolecular interaction (charge-charge and hydrogen bonding)between the methylated ammonium ions (—NH₂(Me)) of N,N′-dimethylL-cystine diamides and the carboxylates (—COO⁻) of L-cystine, so thesemethylated L-cystine diamides cannot bind to the specific cystinecrystal surfaces and, thus, cannot inhibit crystal growth.

FIG. 1 further provides that the five L-cystine diamides, namelyL-cystine dicyclopropylamide (CDCPA, LH704, IV), L-cystine bispiperidine(CDPIP, LH706, VI), L-cystine bismorpholide (CDMOR, LH707, VII),L-cystine bis(N′-methylpiperazide) (CDNMP, LH708, VIII), and L-cystinediethanolamide (CDEOA, LH709, IX), have better activity than the controlcompound CDME and were selected for further characterization. In orderto rank the test compounds, the dose-response curves of the fiveL-cystine diamides was determined in comparison to CDME and calculatedas EC_(2x), the effective concentration required to double thesolubility of L-cystine in water.

FIG. 2 and Table 1, further elaborate that all five L-cystine diamideshave better EC_(2x) than CDME with L-cystine bismorpholide (CDMOR,LH707, VII) and L-cystine bis(N′-methylpiperazide) (CDNMP, LH708, VIII)being the most potent. CDMOR (VII) has an EC₂ about 7-fold lower thanCDME (0.86 vs 3.53 μM) while CDNMP (VIII) is 24-fold more potent thanCDME (EC_(2x) of 0.26 vs 3.53 μM) at increasing the aqueous solubilityof L-cystine.

TABLE 1 Effects of L-cystine diamides on the aqueous solubility ofL-cystine in comparison to CDME EC_(2x) LH# Name Structure (mM)Ratio^(b) Control CDME

6.37 1.0 LH704 CDCPA (IV)

3.53 1.8 LH706 CDPIP (VI)

1.59 4.0 LH707 CDMOR (VII)

0.86 7.4 LH708 CDNMP (VIII)

0.26 24.4 LH709 CDEOA (IX)

2.02 3.2 ^(a)EC_(2x) refers to the concentration required to double theaqueous solubility of cystine. ^(b)Ratio refers to the improvement inpotency over the control CDME.

Based on these studies, the two most effective L-cystine diamideinhibitors are L-cystine bismorpholide (CDMOR, LH707, VII) and L-cystinebis (N′-methylpiperazide) (CDNMP, LH708, VIII); they are 7-24 times moreeffective than CDME in increasing the aqueous solubility of L-cystineand inhibiting cystine crystallization. With these two L-cystinediamides, inhibition of cystine crystallization in vitro occurs atsubmicromolar concentrations, which are much lower than the CDMEconcentration. For such reasons they are ideal candidates for treatingcystinuria to prevent the formation or reduce the rate of the growth ofL-cystine crystals in cystinuria patients.

Example 5 In Vivo Activity in a Genetic Mouse Model Material and MethodsMice

Slc3a1 knockout mice in a mixed 129/C57BL6 background were used in thestudies described here. Two-month old knockout male mice for LH707 orLH708 treatment were selected. Mice at this age exhibit crystaluria butvery few or no bladder stones whereas approximately 50% ofthree-month-old mice exhibit stones, making this the ideal window toassess the effects of treatment. Most male mice over age six months havebladder stones and some have kidney stones as well. Female knockout micehave crystalluria but no stones until age over 12 months. Mousegenotypes and gender were determined by PCR amplification of tail DNA.Based on previous observations, Slc3a1 heterozygotes have no apparentphenotype so they were used in place of wild-type mice as needed. Animalstudies were conducted in accordance with Rutgers University LACUCpolicies.

LH707 or LH708 Administration

LH707 dihydrochloride (MW 451.43) and LH708 tetrahydrochloride (MW550.44) were prepared fresh daily (2.9 mM in water) and 200 μl wasadministered to Slc3a1 knockout male mice (body weight 20 g) by gavagedaily for four weeks using a 20G disposable flexible plastic feedingneedle with a soft tip (Model FTP-20-30, Instech Laboratories, Inc.,Plymouth Meeting, Pa.).

This dose is equivalent to 29 μmol/kg (29 mg/m² assuming a body surfacearea of 0.007 m²). To determine LH707/LH708 absorption and excretion,these compounds were administered daily for seven days and urine samplescollected before and immediately after the last treatment.

Urine Collection and Analysis

Urine samples (0.15 to 1.1 ml) were collected by placing the mice inmetabolic cages for four hours or longer and frozen at −20° C. Thefour-week treatment samples were analyzed for amino acids by ionexchange chromatography on a Biochrom analyzer (Biochrom US, Holliston,Mass.) and the amino acid levels normalized to nmol/mg creatinine. Theseven-day treatment samples were analyzed for LH707 and LH708 by LC-MS(see below).

Micro-Computed Tomography

Mice were sacrificed after urine collection and the bladder and kidneysremoved and placed in 10% formalin. The bladder was scanned ex vivousing a SkyScan 1172 micro CT scanner with a 50 mm field of view (BrukerCorp., Billerica, Mass.). Images were taken with an 11 Mp Hamamatsucamera with a pixel size of 11.51 μm. The source voltage and currentranged between 40-45 kV and 220-250 μA, respectively, and the scan timewas approximately 30 min. The SkyScan reconstruction program NRecon wasused for image reconstruction. The output images were imported into theBruker CT-Analyzer (CTAn) program (version 1.13). This application wasevaluated for the assessment of quantitative parameters such as bladdervolume, stone volume, and stone number from the scanned 3D datasets.

Estimation of Stone Size and Number

After micro CT scanning, bladders were weighed and the stones removed,weighed, counted, and measured in the longest dimension (see FIG. 5).

Analysis of Urine Samples by UPLC-MS

It was determined that the concentrations of LH707/LH708 in the urinesamples collected using LC-MS/MS in positive MRM mode (LH707,379.0→188.9; LH708, 203.1→101.3) on a Transcend LX2 system (ThermoFisher, Waltham, Mass.) coupled to an API 4000 (AB Sciex, Framingham,Mass.). Chromatographic separation was achieved with a HILIC column(2.1×50 mm, 5 rpm, Waters Corporation, Huntingdon Valley, Pa.) using awater (A)/ACN (B) mobile phase system containing 0.1% formic acid (v/v).The gradient was performed at a flow rate of 750 μL/min as follows: 95%B for 90 s, 95 to 5% B in 42 s, 5% B for 30 s.

Data Analysis and Discussion

Fisher's exact test was used to assess the number of mice with andwithout stones in each of the two groups. A two-tailed t-test was usedwith unequal variance to assess differences in bladder weight, stoneweight, and stone number between the LH707/LH708- and water-treatedgroups. Significance level was set at 5%.

Based on the results, inventors concluded that LH708 has a betterefficacy at inhibiting cystine stone formation, as only 1 out 7LH708-treated mice showed stones in their bladder. Further, theconcentration of cysteine in mouse urine was significantly increased ascompared to before treatment. Finally, there were no significant changesin the urine concentration of other amino acids such as ornithine,lysine, arginine, proline, valine, leucine and phenylalanine.

Initially, inventors observed that there was no trace of the testcompounds in urine samples collected prior to oral dosing. However,after the completion of the dosing regimen significant amount of thetest compounds were found in the urine of the participating mice (seeFIG. 6). Existence of micromolar concentrations of LH707 and LH708 ineach mouse after such dosing suggests that the compounds are readilybioavailable when administered orally.

Surprisingly, significantly higher concentrations of LH708 were found incysteineuria mice; the Slc3a1 knockout mice, than in control or normalmice (see FIG. 7). As LH708 has a polyamine core structure, it isconcluded that LH708 may be absorbed similar to polyamines such asspermine or spermidine, rather than to cystine. This is an unexpectedobservation, as it suggests that other transporters (like polyaminetransporters) are likely elevated in cystineuric mice (i.e. afterknocking out the SLC3A1 gene) which worked in favor of LH708availability, but not in LH707.

Finally, the higher volumes of urine collected in LH708-treated mice ascompared to before treatment, as shown in FIG. 8 suggests that LH708could also be having diuretic effects as an additional benefit. As therewere no significant changes in the urine concentration of other aminoacids such as ornithine, lysine, arginine, proline, valine, leucine andphenylalanine, those of ordinary skill in the art can extrapolate thatthe diuretic effects are attributed to the compounds administered and/orthe rise of urine cystine concentration during the treatment course (seeFIG. 9).

While certain of the embodiment of the present invention have beendescribed and specifically exemplified above, it is not intended thatthe invention be limited to such embodiments. Various modifications maybe made thereto without departing from the scope and spirit of thepresent invention as set forth in the following claims. All suchmodifications coming within the scope of the present claims are intendedto be included herein.

1.-8. (canceled)
 9. A prodrug conjugate having the structure of formula(C);

Wherein each R and R′ pair are independently selected from (i) or (ii);(i) R and R′ are independently selected from the group consisting ofhydrogen, substituted or unsubstituted alkyl, substituted orunsubstituted alkenyl, substituted or unsubstituted alcohol, substitutedor unsubstituted aryl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocyclic, and substituted orunsubstituted heteroaryl, or (ii) R and R together form a substituted orunsubstituted heterocyclic ring structure, or a substituted orunsubstituted heteroaryl ring structure; X′ is selected from the groupconsisting of alkoxycarbonyl (ROCO) and (acyloxy)alkoxycarbonyl(RCOOCH(R′)OCO, and R′ is H or alkyl; and Y is O or S.
 10. The conjugateof claim 9, wherein at least one R and R pair together form a ringstructure having the formula (D)

wherein Z′ is CR″—W, N—W or O; and R″ is selected from the groupconsisting of hydrogen, substituted or unsubstituted alkyl, substitutedor unsubstituted alkenyl, substituted or unsubstituted aryl, substitutedor unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl,and substituted or unsubstituted cycloalkenyl; W is selected from thegroup consisting of alkoxycarbonyl (ROCO) and (acyloxy)alkoxycarbonyl(RCOOCH(R′″)OCO; R′″ is H or alkyl, and r is 0-4. 11.-18. (canceled) 19.A method of treating a subject diagnosed with cystinuria comprisingadministering to said subject a pharmaceutically effective amount of acompound having the formula (A):

and pharmaceutically acceptable salts, solvates and prodrugs thereofwherein each R and R′ pair are independently selected from (i) or (ii);(i) R and R′ are independently selected from the group consisting ofhydrogen, substituted or unsubstituted alkyl, substituted orunsubstituted alkenyl, substituted or unsubstituted alcohol, substitutedor unsubstituted aryl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocyclic, and substituted orunsubstituted heteroaryl, or (ii) R and R′ together form a substitutedor unsubstituted heterocyclic ring structure, or a substituted orunsubstituted heteroaryl ring structure; X is hydrogen, or an alkyl; andY is O or S.
 20. The method of claim 19, wherein R and R′ of each pairare independently selected from the group consisting of hydrogen,substituted or unsubstituted alkyl, and substituted or unsubstitutedcycloalkyl.
 21. The method of claim 20, wherein R and R′ of each pairare selected from the group consisting of —CH₃, —CH₂CH₃, —CH₂CH₂OH and—CH(CH₃)₂.
 22. The method of claim 20, wherein R and R of at least onepair together form a ring structure having the formula (B)

wherein Z is CR″-Q, N-Q or O; Q and R″ are independently selected fromthe group consisting of hydrogen, substituted or unsubstituted alkyl,substituted or unsubstituted alkenyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, substituted or unsubstitutedcycloalkyl, and substituted or unsubstituted cycloalkenyl; and r is 0-4.23. The method of claim 22, wherein Z is N-Q; r is 1-3; and Q ishydrogen or an alkyl.
 24. The method of claim 21, wherein said structure(B) is selected from the group consisting of:

and Q is hydrogen or a lower alkyl.
 25. The method of claim 19, whereinthe compound having a structure selected from the group consisting of:


26. The method of claim 25, wherein the compound having a structureselected from the group consisting of:


27. The method of claim 26, wherein the concentration of cysteine insubject's urea is raised by at least 10% after the completion of thetreatment regimen.
 28. A method of synthesizing cysteine diamide analogcomprising the steps of: (i) obtaining N-alkyl cysteine by reducingthiazolidine-4-carboxylic acid; (ii) producing N, N′ dialkyl cysteine byoxidizing the N-alkyl cysteine of step (i) in the presence of catalyticiron (III) chloride; (iii) protecting the secondary amine with Bocanhydride, amidation through activated ester; and (iv) deprotecting theN,N′-dialkyl L-cystine.
 29. The method of claim 28, wherein the cysteinediamide analog has the formula (A):

and pharmaceutically acceptable salts, solvates and prodrugs thereofwherein each R and R′ pair are independently selected from (i) or (ii);(i) R and R′ are independently selected from the group consisting ofhydrogen, substituted or unsubstituted alkyl, substituted orunsubstituted alkenyl, substituted or unsubstituted alcohol, substitutedor unsubstituted aryl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocyclic, and substituted orunsubstituted heteroaryl, or (ii) R and R′ together form a substitutedor unsubstituted heterocyclic ring structure, or a substituted orunsubstituted heteroaryl ring structure; X is hydrogen, or an alkyl; andY is O or S.